This proposal seeks to develop potent and broadly active transcriptional blockades of HIV-1 and novel approaches to modify the genome to abrogate CCR5 expression and deliver therapeutic genes specifically and safely to this site. This submission is a competing renewal for GM065059. In the first funding period of this grant we created novel zinc finger transcription factors, ZF-TFs that target conserved sites in the HIV-1 genome and inhibit viral replication in T cell lines and primary human cells and significantly advanced methods for the development of ZF-TFs. Here we will extend these studies to create a potent cocktail of ZF-TFs that, when delivered, will pose significant challenges to viral escape. We also demonstrated that we could engineer and evolve zinc finger-recombinase fusion proteins (RecZFs) capable of excising a target gene from human cells as well as targeting integration into human cells. Here we aim to further develop this approach to allow us to phenocopy the natural protective 32 mutation in CCR5 through targeted deletion of this gene in primary cells. Furthermore we will advance our studies of RecZFs to enable us to site-specifically integrate our HIV-1 gene blockade expression cassettes into the CCR5 locus, disrupting CCR5 expression. We will address the potential of these approaches to alter the course of HIV-1 infection in T cell lines, primary T cells, and monocytes derived from CD34+ cells. The net result of this study will be an increased understanding of HIV-1 gene regulation, methods to control HIV-1 replication, novel tools to modify the human genome, and a new approach to safely and specifically deliver therapeutic genes. These results will have significant implications for HIV-1 gene therapy and gene therapy in general. Relevance: While highly active anti-retroviral therapy (HAART) has changed the face of HIV-1 disease, viral resistance, toxicity, side-effects of the drug cocktail, cost issues, and patient compliance are problematic for a significant percentage of the affected population We propose to develop a powerful new and long lasting approach to HIV-1 gene therapy that addresses these problems and we hope that its application will significantly improve the treatment of HIV-1 infected individuals. The new approach we develop will be applicable to other diseases.